Project Summary Bone fractures and non-union defects often require surgical intervention where devices are used to correct the defect, and 5-10% of these procedures are compromised by bacterial infection. Current treatment options are limited to sustained, high doses of antibiotics and surgical debridement. These corrective procedures drive up healthcare costs and have sub-optimal patient outcomes as effective antibiotic doses are difficult to attain at the site of the infection due to the presence of a biofilm and toxicity considerations. Furthermore, the emergence of antibiotic-resistant bacteria raises concerns regarding the effectiveness of antibiotics to reduce biomaterial- associated infections. Therefore, there is a significant, unmet need for alternative therapeutic strategies to eliminate device-related infections. The objective of this renewal application is to engineer synthetic hydrogels delivering antimicrobial enzymes to eliminate bacterial infection and promote bone repair. Our central hypothesis is that controlled delivery of lysostaphin from osseo-reparative hydrogels will eliminate Staphylococcal infections and result in bone healing in murine models of implant-associated bone infection. The rationale for this research is that it will establish a localized strategy to effectively reduce bacterial infections during bone healing using a potent antimicrobial protein. Aim 1: Engineer lysostaphin- delivering injectable hydrogels for the treatment of infected bone fractures. We will engineer poly(ethylene glycol) (PEG) hydrogels that release active lysostaphin in response to the local wound environment. We will then test the ability of this material to eliminate Staphylococcal infections and support fracture healing in a pin-stabilized femur fracture model for both prophylactic and established infection scenarios. Aim 2: Engineer PEG hydrogels co-delivering lysostaphin and BMP-2 to eliminate bacterial infection and repair non-healing segmental bone defects. We will evaluate PEG hydrogels co-delivering lysostaphin and BMP-2 in prophylaxis and established infection bone defect cases. The proposed research is innovative because it focuses on engineering new classes of biomaterials that deliver a potent antimicrobial enzyme locally to eliminate bacterial infection and support bone repair. These studies will establish novel bioactive materials that eliminate bone-related infections and enhance bone formation for improved bone repair in various clinical applications.